Bacterial cells are systems of choice for the production of biomolecules, especially of target proteins. Among other advantages, bacterial systems are easy to use and allow the rapid production of large quantities of proteins in a limited volume of culture.
One of the most widely and routinely used bacterial system is the bacteriophage T7 expression system. This bacterial system was described in U.S. Pat. No. 4,952,496. In this system, the gene encoding the target protein is placed under the control of a T7 promoter, and is transformed in a bacterial host, usually E. coli, which comprises an integrated lambda DE3 lysogen phage. The lambda DE3 lysogen phage carries the gene encoding the T7 RNA polymerase under the control of a lacUV5 promoter. When cultured on an IPTG-containing medium, the expression of the T7 RNA polymerase is induced, and allows the expression of the target protein.
Due to the integration of the gene of the T7 RNA polymerase (T7 gene 1) within the sequence of the Int gene, Lambda DE3 phage should be defective in its ability to enter into the lytic phase. However, bacterial lysis is observed during some protein productions and in the absence of any other phage, suggesting that the DE3 phage may recover its lytic properties. The bacterial lysis and even more, the presence of infectious phages in the culture broth is highly problematic because (i) it compromises the use of produced target proteins for some applications, such as, for example, pharmaceutical applications, (ii) the decontamination process in order to remove any trace of phages requires the shutdown of the production lines and the complete renewal of the batches of culture and (iii) it reduces dramatically the yield of recombinant protein.
Alternative methods to the use of a phage have been described:
WO 03/050240 describes an expression system for producing a target protein in a host cell comprising a gene encoding T7 RNA polymerase integrated using homologous recombination. However, the system of WO 03/050240 is difficult to implement, due to the size of the T7 RNA polymerase gene and due to the fact that homologous recombination is not possible or easy to do in all E. coli strains (as mentioned by Phue et al., Biotechnology and Bioengineering, 101, 831-836, 2008). Consequently, the number of transformed cells carrying the T7 RNA polymerase integration remains very low or these cells are not obtained. Moreover, using homologous recombination, it is necessary to use a selective marker to select bacteria containing integration of the T7 RNA polymerase gene. This marker will not be usable for another selection step and could be undesired for the final use of the strain. For example, selective markers often used are antibiotic resistance genes but it is recommended to avoid these genes in biopharmaceutical productions. An additional step is thus required to remove the antibiotic resistance gene from the strain and it is not always possible to do it.
WO 2008/139153 describes another expression system, wherein the host cell is transformed with a plasmid comprising an expression cassette for T7 RNA polymerase.
However, due to the use of a plasmid, hosts cells have to be maintained in selective conditions to make sure that they still comprise the plasmid. In addition, plasmids are frequently subjected to recombination, which impaired the expression system.
Therefore, there is a need for a novel method for producing a biomolecule of interest, wherein, when a phage is used, the culture is not contaminated by infectious phages or unintentionally lysed during growth or protein production.
The present invention hereby provides a genetically modified phage that does not recover its lytic properties during culture, thereby allowing the production of a biomolecule of interest without phage contamination.